The Signal Transduction Protein GlnK Is Required for NifL-Dependent Nitrogen Control of
            <i>nif</i>
            Gene Expression in
            <i>Klebsiella pneumoniae</i>

Jack, Rachael L.; Zamaroczy, Miklos de; Merrick, Mike

doi:10.1128/jb.181.4.1156-1162.1999

Cited by 98 publications

(98 citation statements)

References 57 publications

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“…In the presence of O 2 , however, NifL appears to be oxidized directly by O 2 and dissociates from the membrane [21]. Concerning the nitrogen signal transduction, it is known that uridylylated GlnK transduces the signal of nitrogenlimitation to the nif regulon [8][9][10]21]. Experimental data indicate that under nitrogen-limitation, GlnK interacts with the inhibitory NifL-NifA complex, resulting in the dissociation of the complex (J. Stips and R. A. Schmitz, unpublished observation).…”

Section: Carbon and Energy Sourcementioning

confidence: 99%

Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool

Grabbe

Schmitz

2003

European Journal of Biochemistry

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In Klebsiella pneumoniae, the flavoprotein, NifL regulates NifA mediated transcriptional activation of the N 2 -fixation (nif) genes in response to molecular O 2 and ammonium. We investigated the influence of membrane-bound oxidoreductases on nif-regulation by biochemical analysis of purified NifL and by monitoring NifA-mediated expression of nifH¢-¢lacZ reporter fusions in different mutant backgrounds. NifL-bound FAD-cofactor was reduced by NADH only in the presence of a redox-mediator or inside-out vesicles derived from anaerobically grown K. pneumoniae cells, indicating that in vivo NifL is reduced by electrons derived from membrane-bound oxidoreductases of the anaerobic respiratory chain. This mechanism is further supported by three lines of evidence: First, K. pneumoniae strains carrying null mutations of fdnG or nuoCD showed significantly reduced nif-induction under derepressing conditions, indicating that NifL inhibition of NifA was not relieved in the absence of formate dehydrogenase-N or NADH:ubiquinone oxidoreductase. The same effect was observed in a heterologous Escherichia coli system carrying a ndh null allele (coding for NADH dehydrogenaseII). Second, studying nif-induction in K. pneumoniae revealed that during anaerobic growth in glycerol, under nitrogen-limitation, the presence of the terminal electron acceptor nitrate resulted in a significant decrease of nif-induction. The final line of evidence is that reduced quinone derivatives, dimethylnaphthoquinol and menadiol, are able to transfer electrons to the FAD-moiety of purified NifL. On the basis of these data, we postulate that under anaerobic and nitrogen-limited conditions, NifL inhibition of NifA activity is relieved by reduction of the FAD-cofactor by electrons derived from the reduced quinone pool, generated by anaerobic respiration, that favours membrane association of NifL. We further hypothesize that the quinol/quinone ratio is important for providing the signal to NifL.

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Section: Carbon and Energy Sourcementioning

confidence: 99%

Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool

Grabbe

Schmitz

2003

European Journal of Biochemistry

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“…Both EcGlnK and KpGlnK proteins have high sequence identity to EcPII. However, EcPII expressed from the chromosome is unable to substitute for the GlnKs with respect to NifLA [13,16] . This protein was called GlnK because it has higher identity to EcGlnK than EcPII and it is encoded by a glnK gene which is located on the glnKamtB operon.…”

Section: The T-and C-loopsmentioning

confidence: 99%

“…Jack et al [16] aligned PII and parologue proteins from several organisms and found five residues (positions 3, 5, 52, 54 and 64), which distinguish GlnK from PII proteins. PII proteins contain Lys3, Glu5 or Asp5, Met52 or Val52, Asp54 and Val64.…”

Section: Relationship Of Pii To Glnkmentioning

confidence: 99%

Herbaspirillum seropedicae signal transduction protein PII is structurally similar to the enteric GlnK

Benelli

Buck

Polikarpov

et al. 2002

European Journal of Biochemistry

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PII-like proteins are signal transduction proteins found in bacteria, archaea and eukaryotes. They mediate a variety of cellular responses. A second PII-like protein, called GlnK, has been found in several organisms. In the diazotroph Herbaspirillum seropedicae, PII protein is involved in sensing nitrogen levels and controlling nitrogen fixation genes. In this work, the crystal structure of the unliganded H. seropedicae PII was solved by X-ray diffraction. H. seropedicae PII has a Gly residue, Gly108 preceding Pro109 and the main-chain forms a b turn. The glycine at position 108 allows a bend in the C-terminal main-chain, thereby modifying the surface of the cleft between monomers and potentially changing function. The structure suggests that the C-terminal region of PII proteins may be involved in specificity of function, and nonenteric diazotrophs are found to have the C-terminal consensus XGXDAX(107-112). We are also proposing binding sites for ATP and 2-oxoglutarate based on the structural alignment of PII with PII-ATP/ GlnK-ATP, 5-carboxymethyl-2-hydroxymuconate isomerase and 4-oxalocrotonate tautomerase bound to the inhibitor 2-oxo-3-pentynoate.

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“…PII-like nitrogen sensory proteins have been demonstrated to play a major role in regulating nitrogen metabolism and are considered to be the most widespread signalling proteins in nature [25][26][27]. Recent studies have revealed that the nitrogen status in the diazotrophs K. pneumoniae and Azotobacter vinelandii is transduced towards the regulatory proteins NifL and NifA by the PII-like protein GlnK, a GlnB paralogue [14,17,[28][29][30][31][32]. By contrast to the glnK gene of A. vinelandii, enteric glnK genes are under the control of the general nitrogen regulatory system, and are therefore only expressed under conditions of nitrogen starvation [25,33,34].…”

mentioning

confidence: 99%

“…However, in response to the internal nitrogen status, GlnK is covalently modified by uridylylation, as is GlnB at the conserved tyrosine residue located in the T-loop (Y51), by the uridylyltransferase ⁄ uridylyl-removing enzyme GlnD [25,35,36]. It has been demonstrated that GlnK mediates the cellular nitrogen status towards the NifL ⁄ NifA regulatory system by direct protein interaction; however, the mechanism differs between K. pneumoniae and A. vinelandii [14,17,[28][29][30][31][32]. In A. vinelandii, the nonuridylylated form of GlnK activates the inhibitory function of NifL under nitrogen excess by direct protein-protein interaction [37,38], whereas, under nitrogen limitation, NifA is relieved from NifL inhibition by elevated levels of 2-oxoglutarate binding to the GAFdomain of NifA [4,14,18,32,[37][38][39][40][41].…”

mentioning

confidence: 99%

Towards understanding the nitrogen signal transduction for nif gene expression in Klebsiella pneumoniae

et al. 2008

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In the diazotroph Klebsiella pneumoniae, the nitrogen sensory protein GlnK mediates the cellular nitrogen status towards the NifL/NifA system that regulates transcription of the nitrogen fixation genes in response to ammonium and molecular oxygen. To identify amino acids of GlnK essential for this signal transduction by protein–protein interaction, we performed random point mutagenesis by PCR amplification under conditions of reduced Taq polymerase fidelity. Three thousand two hundred mutated glnK genes were screened to identify those that would no longer complement a K. pneumoniaeΔglnK strain for growth under nitrogen fixing conditions. Twenty‐four candidates resulting in a Nif− phenotype were identified, carrying 1–11 amino acid changes in GlnK. Based on these findings, as well as structural data, several single mutations were introduced into glnK by site‐directed mutagenesis, and the Nif phenotype and the respective effects on NifA‐mediated nif gene induction was monitored in K. pneumoniae using a chromosomal nifK′–′lacZ fusion. Single amino acid changes resulting in significant nif gene inhibition under nitrogen limiting conditions were located within the highly conserved T‐loop (A43G, A49T and N54D), the body of the protein (G87V and K79E) and in the C‐terminal region (I100M, R103S, E106Q and D108G). Complex formation analyses between GlnK (wild‐type or derivatives) and NifL or NifA in response to 2‐oxoglutarate indicated that: (a) besides the T‐loop, the C‐terminal region of GlnK is essential for the interaction with NifL and NifA and (b) GlnK binds both proteins in the absence of 2‐oxoglutarate, whereas, in the presence of 2‐oxoglutarate, NifA is released but NifL remains bound to GlnK.

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The Signal Transduction Protein GlnK Is Required for NifL-Dependent Nitrogen Control of nif Gene Expression in Klebsiella pneumoniae

Cited by 98 publications

References 57 publications

Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool

Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool

Herbaspirillum seropedicae signal transduction protein PII is structurally similar to the enteric GlnK

Towards understanding the nitrogen signal transduction for nif gene expression in Klebsiella pneumoniae

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